ABSTRACT: Uterine leiomyoma (fibroids) are benign tumors that develop during reproductive age, and symptomatic tumors account for 1/3 of hysterectomies performed in the United States. Although the molecular mechanisms that cause formation and development of leiomyoma are unknown, leiomyoma cells overexpress pro-inflammatory and pro-fibrotic genes, suggesting that inflammation and fibrosis are key drivers of their growth and development. If so, then it could be possible to develop non-surgical therapeutic approaches to prevent, manage, or reverse leiomyoma development, but designing such strategies will require a deeper knowledge of underlying molecular mechanisms. Expression profiling has identified many miRNAs with altered expression in leiomyoma as compared to myometrium, and our laboratory has focused on understanding how these miRNAs impact pro-inflammatory and pro-fibrotic pathways and effectors in leiomyoma. Our results strongly implicate two miRNAs?miR-200c and miR-29c?in the development of leiomyoma. Expression of both miR-200c and miR-29c is significantly reduced in leiomyoma compared to adjacent normal myometrium. We showed that miR-200c regulates expression of IL-8 in leiomyoma smooth muscle cells (LSMC) by altering NF-kB activity and by directly targeting IKBKB, whose phosphorylated form was elevated in leiomyoma as compared to matched myometrium, collectively suggesting enhanced NF-kB signaling in leiomyoma. Based on our published and preliminary results we hypothesize that a co-regulatory interaction between NF-kB signaling and epigenetic modification serves as the underlying mechanism that promotes miR-200c and miR-29c suppression, resulting in elevated expression of their specific target genes whose products play a central role in pathogenesis of leiomyoma. This central hypothesis will be tested in two specific aims. Aim 1 will determine the mechanism underlying miR-200c and miR-29c suppression in leiomyoma. We hypothesize that the low level of expression of miR-200c/miR-29c in leiomyomas is due to activation of NF-kB signaling leading to their transcriptional repression by NF-kB (p65) and to epigenetic modification through DNMT1, DNMT3A and DNMT3B, which are also regulated by NF-kB signaling. Aim 2 will determine the therapeutic potential of an NF-kB signaling inhibitor (Bay 11-7082), a DNA methyltransferase inhibitor (Zebularine) and anti- inflammatory/anti-fibrotic agent (Tranilast) on leiomyoma initiation and regression in a mouse leiomyoma model. The proposed interactive specific aims through novel mechanistic approaches will decipher the independent and co-regulatory functions of miRNAs and NF-kB signaling pathway that lead to leiomyoma growth, and will determine the potential utility of novel therapeutic drugs that influence the expression of these miRNAs and could be useful as a pharmacologic treatment of leiomyomas. If so, this could reduce the number of relatively costly hysterectomies performed, reduce or eradicate symptoms of leiomyoma, and preserve fertility in women with leiomyoma who may still desire to bear children.